Fluorescence is a physical phenomenon based upon the ability of certain molecules to absorb and emit light at different wavelengths. The absorption of light (photons) at a first wavelength is followed by the emission of photons at a second wavelength and different energy state. If the emission is relatively short-lived, i.e., approximately 10.sup.-8 seconds, it is referred to as "fluorescence". "Fluorophores" are substances which release significant amounts of fluorescent light.
The fluorescence of many fluorophores is dependent upon the pH of their environment. Therefore, fluorescence of a particular fluorophore is typically measured in the pH region associated with its maximum intensity. See, for example, U.S. Pat. No. 4,774,339 which is incorporated herein by reference.
Fluorophores can be segregated into two broad classes: "intrinsic fluorescent substances" and "extrinsic fluorescent substances". Intrinsic fluorophores comprise naturally occurring biological molecules whose ability to absorb exciting light and emit fluorescent light is based directly on their internal structure and chemical formulation. Extrinsic fluorophores do not occur naturally; they are developed or created in the laboratory.
In order to be useful in medical diagnostics, e.g., immunoassays, the fluorophore: (1) should be capable of being tightly bound on a chemical entity; (2) should be sensitive to those changes in the environmental test conditions or systems indicative of chemical change; and (3) should only minimally affect the features or properties of the molecule being investigated. Investigators typically utilize one of two approaches to bind the fluorophore to a ligand or the analyte of interest: (1) direct binding; or (2) chemically combining the fluorophore with another composition which, in turn, has the requisite specific binding capacity to the ligand to form a conjugate molecule. In the latter, the binding specificity of the conjugate is provided by the other compound and the light emitting capacity is provided by the fluorophore.
When a fluorophore is excited by a plane-polarized beam of light, the molecule will emit a polarized beam of light. The degree of polarization can be determined by the equation ##EQU1## where: IV is the light intensity from the sample when excited by vertically polarized light and IH is the light intensity from the sample when excited by horizontally polarized light.
The degree of polarization can be used to determine the concentration of a particular analyte in a sample. For example, in an immunoassay, an analyte-fluorophore complex will have a low degree of polarization compared to a complex of a binding partner for the analyte, the analyte and the fluorophore. This is because the analyte-fluorophore complex, referred to herein as a "tracer," is smaller and thus has more random rotation in the time between its absorption of excitation light and the emission of fluorescence. Upon addition of the binding partner, however, the rotation of the bound-tracer is less random because of the greater mass of the compound. Thus, the degree of polarization for the bound tracer increases relative to that of the unbound tracer because the rotation of the bound tracer has decreased relative to the rotation of the unbound tracer. This inverse relationship between degree of polarization and rotation is the basis of fluorescence polarization immunoassay ("FPI") techniques.
FPI can be utilized to determine the concentration of an analyte in a sample containing or suspected of containing the analyte, such as, the concentration of an antigen, antibody, hapten, therapeutic drug or drug of abuse (or the metabolic products of a therapeutic drug or drug of abuse) in a bodily fluid. For example, if a drug of abuse is the analyte of interest to be measured, a known quantity of the drug is labeled with a fluorophore, such as fluorescein. The resultant drug-fluorescein complex is the tracer. The tracer and a specific binding partner for the drug can then be introduced into a patient sample suspected of including the drug. The tracer and any non-labelled drug in the sample will then compete for the limited number of binding sites on the specific binding partner. Each will have an equal probability of complexing with the specific binding partner. The observed polarization of fluorescence of the tracer becomes a value somewhere between that of the free and bound tracer. If the patient sample contains a high concentration of the drug, the observed polarization value will be low. This is because there is more of the drug in the sample than tracer and, as such, more of the drug from the sample will bind to the binding partner than will tracer. The tracer will remain relatively free in solution and, upon excitation by plane-polarized light, will maintain a relatively random rotation. However, if the patient sample contains a low concentration of the drug, the observed polarization value will be high because most of the tracer will be bound to the binding partner. Thus, the amount of the drug present in a sample is inversely proportional to the observed degree of polarization.
By sequentially exciting the reaction mixture of an immunoassay with vertically and then horizontally polarized light and analyzing only the vertical component of the emitted light, the polarization of fluorescence in the reaction container can be determined very accurately. The degree of polarization can be calculated from Equation 1, and the precise relationship between polarization and concentration of the unlabelled drug is established by measuring the polarization values of calibrators containing known concentrations of the drug. Alternatively, the reaction mixture of an immunoassay can be excited with vertically polarized light followed by analyzing, alternately, the horizontal and vertical component of the emitted light.
An inherent problem of FPI is light scattering. Light scattering may produce either an increase or a decrease in the apparent fluorescence signal. If the fluorescence value is artificially increased or decreased, then the concentration attributed to the analyte of interest will be similarly skewed. If the concentration of the analyte to be measured is necessary for diagnostic purposes, such skewed results can have severe and unacceptable consequences.
Previous light scattering correction methods involve addition of a buffer to a transparent container or cuvette, followed by addition of up to half of the test sample. A light sample intensity, or "scatter-correction" reading is then taken; this first reading is attributed to light scatter caused by the sample. Thereafter, a fluorophore is added to the sample, and after an appropriate incubation period, a second light intensity reading is taken. In order to determine a scatter corrected fluorescence reading due solely to the interaction of the fluorophore and analyte of interest, the scatter correction reading must be subtracted from the second light intensity reading. Thus, a primary disadvantage with this method is that it requires additional time to obtain the scatter-correction reading.
Because fluorescence is a valuable tool in the determination of the concentration of species of interest in a test sample, it would be useful to obtain a fluorescence value independent of light scattering.